青海湖裸鲤幼鱼热激蛋白60基因克隆及其在碳酸盐碱度胁迫下的表达

Molecular characterization of GpHsp60 from juvenile Gymnocypris przewalskii:Effects of alkalinity on its expression

通过青海湖裸鲤(Gymnocypris przewalskii)转录组数据获得热激蛋白60基因(GpHsp60)的序列片段,利用RACE克隆完善GpHsp60的mRNA全序列,得到序列全长2995 bp:包括开放阅读框1728 bp,5′UTR和3′UTR分别为137 bp和113 bp;编码氨基酸575个,分子量为61.34 kDa,理论等电点为5.38,具有线粒体Hsp60高度保守序列。通过实时荧光定量PCR检测发现,青海湖裸鲤幼鱼受碳酸盐碱度(29 mmol·L^(-1))胁迫后,GpHsp60基因在脑、鳃、肝、肾、心脏、肠、肌肉、脾脏和皮肤等组织都有所表达。与对照组相比,胁迫组青海湖裸鲤幼鱼鳃(1.99倍)、脑(1.95倍)、肝脏(3.52倍)、心脏(2.29倍)、肌肉(3.53倍)和肾脏(4.34倍)中GpHsp60基因都呈现高表达,说明以上组织细胞中的Hsp60可能被招募参与应答水环境碱度胁迫;而肠(0.257倍)和脾脏(0.251倍)组织中的Hsp60表达量却呈现下降,这可能与组织细胞线粒体功能效率降低有关;皮肤组织在受碱度胁迫后GpHsp60基因表达量无显著变化,表明青海湖裸鲤幼鱼皮肤组织未参与对碳酸盐碱度胁迫的应答。研究结果为揭示Hsp60基因在青海湖高碳酸盐碱度环境中生活的特有物种青海湖裸鲤在碱度胁迫下发挥的作用,为探讨和研究高碳酸盐碱度胁迫下水生生物体内分子响应机制研究提供了基础数据。

Heat shock protein is a kind of stress reactive protein produced by organisms under physical,chemical,biological and other stimulation conditions.In order to get the whole sequences of heat shock protein 60 gene(Hsp60),the transcriptome data of Gymnocypris przewalskii kidney tissue was downloaded from NCBI database.We obtained the sequence fragments of Hsp60 by alignment from assembled and spliced transcriptome data.The nested primers were designed and RACE technology was utilized to acquire the whole Hsp60 of Gymnocypris przewalskii(GpHsp60)mRNA sequence.The above sequence fragments were spliced together by bioinformatics analysis.The full length of GpHsp60 was 2995 bp,including 1728 bp open reading frame(ORF),5′UTR and 3′UTR for 137 bp and 113 bp,respectively.The online analysis software ProtParam predicted that GpHsp60 gene encoded a protein with the molecular weight of 61.34 kDa,containing 575 amino acids.The theoretical isoelectric point of GpHsp60 was 5.38.Acute carbonate alkalinity(CA)stress experiment was conducted on Gymnocypris przewalskii juveniles to test the expression of GpHsp60 gene.Real time fluorescence quantitative PCR results showed that GpHsp60 gene was expressed in all test tissues,including brain,gill,liver,kidney,heart,intestine,muscle,spleen and skin in G.przewalskii juvenile fish under the stress of carbonate alkalinity(29 mmol·L^(-1)).Compared with the control group,the transcription level of GpHsp60 in juveniles of G.przewalskii showed higher expressions in gill(1.99 times),brain(1.95 times),liver(3.52 times),heart(2.29 times),muscle(3.53 times)and kidney(4.34 times)in stress group.And the elevated expression occurred ranging from 1 to 3 hours in these tissues after stress.And 6 to 9 hours after stress,GpHsp60 expression level returned to control level.The phenomenon indicated that GpHsp60 gene was involved in physiological responses and detoxification process in G.przewalskii juveniles when they swam from freshwater to the waters with high carbonate alkalinity,which meant that the related molecular pathways was activated to respond to carbonate alkalinity stress.However,GpHsp60 expression in the intestine(0.257 time)and spleen(0.251 time)decreased.That might be related to the reason that stress responses in intestine and spleen were weaker and mitochondrial function efficiency of tissue cells was also reduced after high carbonate alkalinity stress.It was suggested that G.przewalskii juveniles mainly provided energy to tissues for functions such as ion regulation,acid-base balance and motor system in the initial stage after entering the high carbonate alkaline environment.While the import to functions such as digestion and blood circulation was reduced,so as to adapt to the new environment quickly.There was no significant variation in GpHsp60 gene expression in skin with the function of water resistance and ion blocking under alkalinity stress.